interpretation of gram stains and other common microbiologic slide preparations - library

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Interpretation of Gram Stains and Other Common Microbiologic Slide Preparations

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Created: Tuesday, 06 February 2007

Last update: Friday, 19 August 2011

AuthorFred Tenover

Author - SecondaryJ. V. Hirschmann

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Interpretation of Gram Stain & Other Microscopic Slides (Enlarged view)

InformationWith contributions by:

Ellen Jo Baron

Larry Carlson

Carla Clausen

Sharon Hilliar

Greg Raugi

Michael Rinaldi

Jane Schwebke

Edited and revised by Rebecca Buxton (corresponding author)

Department of Pathology

University of Utah

Salt Lake City, UT 84132 USA

Email: [email protected]

Originally published in 1990 as Interpretation of Gram Stains and Other Common Microbiologic Slide Preparations by the UpJohn Company,

Kalamazoo, Mich. Permission granted to the ASM MicrobeLibrary by Pfizer Inc.

Introduction

This Atlas was written to help clinicians, microbiologists, and laboratory personnel identify organisms in infected materials stained by techniques

commonly used in most clinical laboratories. Because of its t ime-honored value as a teaching tool for clinical laboratorians and physicians, as much

of the Atlas' original print format as possible has been preserved for this electronic version, with some revision in organism nomenclature.

Since microbes grown on culture media may look different from those in the original clinical specimens, only stains of clinical specimens are

included. Below find a brief description of the stains used and of microscopic techniques, followed by links to sections of images and descriptions of

the pathogens recovered from the following body sources: sputum; urine; vaginal, cervical and urethral secretions; skin; intraabdominal abscesses;

spinal fluid; and miscellaneous tissues.

Suspicion that a patient has an infection arises from the clinician’s interpretation of the history, the physical examination, certain laboratory test

results, and, sometimes, radiographic procedures. The definitive diagnosis typically depends on isolating pathogens on appropriate culture media.

However, many patients require prompt treatment before the results of the culture are available. Frequently, presumptive identification of the

pathogens can be made from microscopic examination of infected material (e.g., sputum, pus, urine, cerebrospinal fluid), and therapy can be chosen

confidently and rationally. Since bacteria cause so many serious treatable infections, this collection focuses on the Gram stain, the most important

staining technique for identifying bacteria using light microscopy. However, several other microbiologic slide preparations are also discussed and

illustrated.

Unless otherwise noted, each micrograph was taken through an oil-immersion (100x) lens (1,000x total magnification).

Identification of bacteria by Gram and acid-fast stains

Because bacterial protoplasm is acidic, a characteristic caused largely by nucleic acids, intact bacteria stain readily with alkaline dyes such as crystal

violet, methylene blue, and basic fuchsin, but stain poorly with acidic dyes such as eosin. The most useful microscopic techniques for identifying

bacteria are differential stains that employ two or more alkaline dyes to distinguish types of bacteria according to the differences in the chemical

composition of their cell walls. The two most important are the Gram stain and the acid-fast stain.

The Gram stain was originally devised in 1884 by Hans Christian Gram (1853–1938), a Danish physician, to detect bacteria in diseased tissue. For a

procedure see the MicrobeLibrary Gram Stain Protocol section (please note you need a subscription to access the protocol). Most bacteria stain

either blue (gram positive) or red (gram negative). In both types, crystal violet and iodine form a complex within cells. However, the cell walls of

gram-positive organisms have a thick layer of peptidoglycan nearly impermeable to the decolorizing agent, and the blue complex of dyes remains

imprisoned within the cell walls. The cell walls of gram-negative bacteria have a thin layer of peptidoglycan and a high lipid concentration that

allows alcohol to penetrate and leach out the crystal violet-iodine complex. Those organisms then take up the red counterstain.

Certain bacteria, especially mycobacteria, have such high lipid concentrations in their cell walls that the organisms stain only feebly with the Gram

technique. They are readily visible, however, with acid-fast stains, such as Ziehl-Neelsen or auramine O. With the Ziehl-Neelsen technique those

organisms take up the red primary stain, carbol fuschin; unlike other bacteria, their cell walls are relatively impermeable to acid-alcohol and also to

the counterstain, methylene blue. Acid-fast organisms, therefore, stain red; the background and other bacteria stain blue. Acid-fast organisms also

take up auramine O, a fluorescent microscopy stain that appears yellow on a dark green to black background.

Some bacteria do not readily incorporate the dyes within their cells and are not easily detected by either Gram or acid-fast stains. Some fungi are

visible when stained with Gram stain, but other microorganisms, such as mycoplasmas, rickettsiae, chlamydiae, and viruses, do not take up the dyes

or are too small to be seen with light microscopy.

To be visible on a slide, organisms that stain by the Gram method must be present in concentrations of about 104 to 105organisms per milliliter of

uncentrifuged fluid. At lower concentrations, the Gram stain of a clinical specimen seldom reveals organisms even if the culture is positive. For

example, Gram stains of ascetic fluid from patients with spontaneous bacterial peritonitis are commonly negative despite positive cultures, because

the number of bacteria is usually less than 10 per milliliter.



Examination of Gram stains

The examiner should first determine whether the stain is adequate (see Sputum–Unacceptable Specimens and Staining Artifacts).In an appropriately

stained specimen, the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is insufficient. In thick specimens like sputum, if certain

areas are difficult to decolorize, the examiner should look for other areas on the slide where the nuclei are red. If the entire specimen is

unsatisfactory, it should be decolorized again with alcohol or acetone-alcohol and then restained with safranin. Repeating the crystal violet and

iodine staining is unnecessary. If the decolorization is still inadequate, the slide should be flooded with acid-alcohol (used in acid-fast stains) to

remove all stain, and the complete Gram stain procedure should be repeated. If the entire specimen is too thick for adequate decolorization, a new

slide with a thinner smear must be prepared.

If both red and blue organisms are visible, decolorization is satisfactory. Excessive decolorization will cause gram-positive organisms to appear to be

gram negative. If only gram-negative organisms are visible and their morphology suggests that they are really gram positive, the entire staining

procedure should be repeated. You may refer to the "Comments and T ips" section of the Gram Stain Protocol for further suggestions (please note

you need a subscription to access the protocol).

When examining a properly stained slide, the examiner should note the following characteristics:

the presence of a single type or several types of organisms

the predominant type of organism if more than one is present

the staining characteristics (gram positive or gram negative)

the shape of the organisms, rods (bacilli) or round (cocci)

the size of the organisms: small, large, thin, plump

the configuration: single organisms, pairs, chains, clumps, clusters, branching

the relation to inflammatory cells because some organisms are characteristically inside inflammatory cells (intracellular) or adherent to

them

With that information, the source of the specimen, and a knowledge of the organisms likely to cause infections at the involved site, the examiner

can presumptively identify many of the pathogens. However, the genus or species of organisms cannot be predicted reliably from their appearance

on a Gram stain. In the case of a patient with acute pneumonia, for example, a properly collected sputum specimen that demonstrates mostly

lancet-shaped, gram-positive diplococci will probably grow Streptococcus pneumoniae (pneumoccocci). Although other streptococci can look

identical on a Gram stain, pneumococcal pneumonia can be diagnosed confidently because other morphologically similar streptococci only rarely

cause acute pulmonary infections. Similarly, to differentiate enteric gram-negative bacilli by their microscopic appearance is difficult . Plump, gram-

negative rods in a sputum specimen from a patient with pneumonia strongly suggest that an enteric gram-negative bacillus is the cause. However it

cannot be safely assumed to be Klebsiella pneumoniae instead of other enteric bacilli such as Escherichia coli, Enterobacter species, or Serratia

marcescens,because these bacteria can also cause lower respiratory tract infections. Clinicians using the Gram stain to help make therapeutic

decisions must recognize the limitations of the technique and not overinterpret the findings.

Links to photographs and legends:

Examination of Gram Stains of Sputum

Examination of Gram Stains of Urine (6 images)

Examination of Gram Stains of Vaginal Secretions (6 images)

Examination of Gram Stains of Cervical and Urethral Discharges (3 images)

Examination of Gram Stains of Bacterial Skin Infections

Examination of Gram Stains of Intraabdominal Infections (5 images)

Examination of Gram Stains of Spinal Fluid–Bacterial Meningitis

Examination of Gram Stains of Miscellaneous T issue Infections (6 images)

Alphabetical Index of Organisms for Interpretation of Gram Stain and Other Common Microbiologic Slide Preparations

References.

Primary reference:

1. Tenover, F. C., and J. V. Hirschmann. 1990. Interpretation of Gram stains and other common microbiologic slide preparations. The UpJohn

Company, Kalamazoo, Mich.

References for editing and revisions in nomenclature:

2. Kwon-Chung, K. J., and J. E. Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia, Pa.

3. Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken. 2003. Manual of clinical microbiology. ASM Press,

Washington, D.C.

Related ContentGram Stain ProtocolsExamination of Gram Stains of Cervical and Urethral DischargesExamination of Gram Stains of Intraabdominal Infections

Sputum–Unacceptable Specimens and Staining Artifacts



Examination of Gram Stains of SputumExamination of Gram Stains of Vaginal SecretionsExamination of Gram Stains of Bacterial Skin InfectionsExamination of Gram Stains of Spinal Fluid—Bacterial MeningitisExamination of Gram Stains of Miscellaneous Tissue InfectionsExamination of Gram Stains of Urine

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Tags: Cell biology (248) , Microbial genetics (74) , Microbes in humans (371)

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interpretation of gram stains and other common microbiologic slide preparations - library

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